Methods of using 4-(amino)-2-(2,6-dioxo(3-piperidly))-isoindoline-3-dione for the treatment of myelodysplastic syndromes

ABSTRACT

Methods of treating, preventing and/or managing myelodysplastic syndromes are disclosed. Specific methods encompass the administration of an immunomodulatory compound, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, alone or in combination with a second active ingredient, and/or the transplantation of blood or cells. Specific second active ingredients are capable of affecting or blood cell production. Pharmaceutical compositions, single unit dosage forms, and kits suitable for use in methods of the invention are also disclosed.

This application is a continuation application of U.S. patentapplication Ser. No. 11/654,550, filed Jan. 16, 2007, now U.S. Pat. No.7,393,863, which is a divisional application of Ser. No. 10/411,649, nowU.S. Pat. No. 7,189,740, which claims priority to U.S. provisionalapplication No. 60/418,468 filed on Oct. 15, 2002, the entirety of whichis incorporated herein by reference.

1. FIELD OF THE INVENTION

This invention relates to methods of treating, preventing and/ormanaging myelodysplastic and related syndromes which comprise theadministration of immunomodulatory compounds alone or in combinationwith known therapeutics. The invention also relates to pharmaceuticalcompositions and dosing regimens. In particular, the inventionencompasses the use of immunomodulatory compounds in conjunction withtransplantation therapy and/or other standard therapies formyelodysplastic syndromes.

2. BACKGROUND OF THE INVENTION

2.1. Pathobiology of MDS

Myelodysplastic syndrome (“MDS”) refers to a diverse group ofhematopoietic stem cell disorders. MDS is characterized by a cellularmarrow with impaired morphology and maturation (dysmyelopoiesis),peripheral blood cytopenias, and a variable risk of progression to acuteleukemia, resulting from ineffective blood cell production. The MerckManual 953 (17^(th) ed. 1999) and List et al., 1990, J. Clin. Oncol.8:1424.

The initial hematopoietic stem cell injury can be from causes such as,but not limited to, cytotoxic chemotherapy, radiation, virus, chemicalexposure, and genetic predisposition. A clonal mutation predominatesover bone marrow, suppressing healthy stem cells. In the early stages ofMDS, the main cause of cytopenias is increased programmed cell death(apoptosis). As the disease progresses and converts into leukemia, genemutation rarely occurs and a proliferation of leukemic cells overwhelmsthe healthy marrow. The disease course differs, with some cases behavingas an indolent disease and others behaving aggressively with a veryshort clinical course that converts into an acute form of leukemia.

The actual incidence of MDS in the U.S. is unknown. MDS was firstconsidered a distinct disease in 1976, and occurrence was estimated at1500 new cases every year. At that time, only patients with less thanfive percent blasts were considered to have this disorder. Statisticsfrom 1999 estimated 13,000 new cases per year and about 1000 cases peryear in children, surpassing chronic lymphocytic leukemia as the mostcommon form of leukemia in the western hemisphere. The perception thatthe incidence is increasing may be due to improvements in recognitionand criteria for diagnosis. The disease is found worldwide.

An international group of hematologists, the French-American-British(FAB) Cooperative Group, classified MDS disorders into five subgroups,differentiating them from acute myeloid leukemia. The Merck Manual 954(17^(th) ed. 1999); Bennett J. M., et al., Ann. Intern. Med. 1985 Oct.,103(4): 620-5; and Besa E. C., Med. Clin. North Am. 1992 May, 76(3):599-617. An underlying trilineage dysplastic change in the bone marrowcells of the patients is found in all subtypes.

There are two subgroups of refractory anemia characterized by fivepercent or less myeloblasts in bone marrow: (1) refractory anemia (RA)and; (2) RA with ringed sideroblasts (RARS), defined morphologically ashaving 15% erythroid cells with abnormal ringed sideroblasts, reflectingan abnormal iron accumulation in the mitochondria. Both have a prolongedclinical course and low incidence of progression to acute leukemia. BesaE. C., Med. Clin. North Am. 1992 May, 76(3): 599-617.

There are two subgroups of refractory anemias with greater than fivepercent myeloblasts: (1) RA with excess blasts (RAEB), defined as 6-20%myeloblasts, and (2) RAEB in transformation (RAEB-T), with 21-30%myeloblasts. The higher the percentage of myeloblasts, the shorter theclinical course and the closer the disease is to acute myelogenousleukemia. Patient transition from early to more advanced stagesindicates that these subtypes are merely stages of disease rather thandistinct entities. Elderly patients with MDS with trilineage dysplasiaand greater than 30% myeloblasts who progress to acute leukemia areoften considered to have a poor prognosis because their response rate tochemotherapy is lower than de novo acute myeloid leukemia patients. TheWorld Health Organization (WHO) classification (1999) proposes toinclude all cases of RAEB-T, or patients with greater than 20%myeloblasts, in the category of acute leukemia because these patientshave similar prognostic outcomes. However, their response to therapy isworse than the de novo or more typical acute myelogenous leukemia oracute nonlymphocytic leukemia (ANLL) patient. Id.

The fifth type of MDS, the most difficult to classify, is called chronicmyelomonocytic leukemia (CMML). This subtype can have any percentage ofmyeloblasts but presents with a monocytosis of 1000/dL or more. It maybe associated with splenomegaly. This subtype overlaps with amyeloproliferative disorder and may have an intermediate clinicalcourse. It is differentiated from the classic chronic myelocyticleukemia (CML) that is characterized by a negative Ph chromosome. Therecent WHO classification (1999) proposes that juvenile andproliferative CMML be listed separately from FAB underMDS/myeloproliferative disorder (MPD) with splenomegaly and greater than13,000 total WBC. CMML is limited to monocytosis, less than 13,000/mm³total leukocytes, and requires trilineage dysplasia. Id. Harris N. L.,et al., J. Clin. Oncol. 1999 Dec., 17(12): 3835-49. Finally, some otherinternational organizations, including WHO, have suggested a sixth classof MDS patients, characterized by a del (5q) abnormality.

MDS is primarily a disease of elderly people, with the median onset inthe seventh decade of life. The median age of these patients is 65years, with ages ranging from the early third decade of life to as oldas 80 years or older. The syndrome may occur in any age group, includingthe pediatric population. Patients who survive malignancy treatment withalkylating agents, with or without radiotherapy, have a high incidenceof developing MDS or secondary acute leukemia. About 60-70% of patientsdo not have an obvious exposure or cause for MDS, and are classified asprimary MDS patients.

The most common cases of MDS are primary, or idiopathic. However, anonspecific history of exposure to indeterminable chemicals or radiation10-15 years prior to onset of disease may be present in about 50% ofpatients. This relationship to pathogenesis remains unproved. Compoundssuch as, but not limited to, benzene, insecticides, weed killers, andfungicides are possible causes of MDS. Goldberg H., et al., Cancer Res.1990 Nov. 1; 50(21): 6876-81. Secondary MDS describes development of MDSor acute leukemia after known exposures to chemotherapy drugs that cancause bone marrow damage. These drugs are associated with a highincidence of chromosomal abnormalities following exposure and at thetime of MDS or acute leukemia diagnosis.

Further, MDS is associated with complications associated with severecytopenias. Other complications are development of myelofibrosis, whichcan accelerate decline in blood counts and increase transfusionrequirements. Transformation to acute leukemia accelerates thedevelopment of complications such as anemia, bleeding, and infections.

Recently, the International MDS Risk Analysis (IMRA) Workshop proposedan International Prognosis Scoring System (IPSS) to decrease imprecisionin predicting survival and AML risk in MDS patients. The IPSS is basedon the number of cytopenias, percentage of BM blasts, and type ofcytogenetic abnormalities (Table 1). Greenberg et al., Blood 1997,89:2079-88. The latter are categorized into good (normal, −Y, del (5q),del (20q)), intermediate, and poor subgroups (complex or chromosome 7abnormalities).

TABLE 1 International Prognostic Scoring System for MDS Prognostic ScoreValue Variable 0 0.5 1.0 1.5 2.0 Bone marrow <5 5-10 — 11-20 21-30blasts (%) Karyotype* Good Intermediate Poor Cytopenias 0/1 2/3 *Good,normal, del (5q), del (20q), −Y; Poor, complex (>3) or chromosome 7abnormalities; Intermediate, +8, and other single or doubleabnormalities.

2.2. MDS Treatment

The current treatment of MDS is based on the stage and the mechanism ofthe disease that predominates the particular phase of the diseaseprocess. Bone marrow transplantation has been used in patients with poorprognosis or late-stage MDS. Epstein and Slease, 1985, Surg. Ann.17:125. This type of therapy, however, is both painful for donor andrecipient, because of the involvement of invasive procedures and cancause severe and even fatal complications to the recipient, particularlywith allogeneic transplant and related Graft Versus Host Disease (GVHD)results. Therefore, the risk of GVHD restricts the use of bone marrowtransplantation to patients with otherwise fatal diseases. Further, asmost patients are elderly and only a few young MDS patients will have amatched donor, the use of bone marrow transplantation is limited.

An alternative approach to therapy for MDS is the use of hematopoieticgrowth factors or cytokines to stimulate blood cell development in arecipient. Dexter, 1987, J. Cell Sci. 88:1; Moore, 1991, Annu. Rev.Immunol. 9:159; and Besa E. C., Med. Clin. North Am. 1992 May, 76(3):599-617. The process of blood cell formation, by which a small number ofself-renewing stem cells give rise to lineage specific progenitor cellsthat subsequently undergo proliferation and differentiation to producethe mature circulating blood cells has been shown to be at least in partregulated by specific hormones. These hormones are collectively known ashematopoietic growth factors. Metcalf, 1985, Science 229:16; Dexter,1987, J. Cell Sci. 88: 1; Golde and Gasson, 1988, Scientific American,July: 62; Tabbara and Robinson, 1991, Anti-Cancer Res. 11:81; Ogawa,1989, Environ. Health Presp. 80:199; and Dexter, 1989, Br. Med. Bull.45:337. The most well characterized growth factors includeerythropoietin (EPO), granulocyte macrophage colony stimulating factor(GM-CSF), and granulocyte colony stimulating factor (G-CSF). Apart frominducing proliferation and differentiation of hematopoietic progenitorcells, such cytokines have also been shown to activate a number offunctions of mature blood cells, including influencing the migration ofmature hematopoietic cells. Stanley et al., 1976, J. Exp. Med. 143:631;Schrader et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:323; Moore etal., 1980, J. Immunol. 125:1302; Kurland et al., 1979, Proc. Natl. Acad.Sci. U.S.A. 76:2326; Handman and Burgess, 1979, J. Immunol. 122:1134;Vadas et al., 1983, Blood 61:1232; Vadas et al., 1983, J. Immunol.130:795; and Weibart et al., 1986, J. Immunol. 137:3584.

Unfortunately, hematopoietic growth factors have not proven effective inmany clinical settings. Clinical trials of MDS patients treated withrecombinant human GM-CSF and G-CSF have shown that while these cytokinescan restore granulocytopoiesis in treated patients, their efficacy isrestricted to the granulocyte or monocyte lineage with little or noimprovement in hemoglobin or platelet counts. Schuster et al., 1990,Blood 76 (Suppl.1):318a. When such patients were treated withrecombinant human EPO, a sustained improvement in hemoglobin or decreasein transfusion requirement was achieved in only less than 25% ofpatients. Besa et al., 1990, 76 (Suppl.1): 133a; Hellstrom et al., 1990,76 (Suppl.1):279a; Bowen et al., 1991, Br. J. Haematol. 77:419.Therefore, there remains a need for safe and effective methods oftreating and managing MDS.

2.3. Thalidomide and Other Compounds useful in the Treatment of Disease

Thalidomide is a racemic compound sold under the tradename Thalomid® andchemically named α-(N-phthalimido)glutarimide or2-(2,6-dioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dione. Thalidomide wasoriginally developed in the 1950's to treat morning sickness, but due toits teratogenic effects was withdrawn from use. Thalidomide has beenapproved in the United States for the acute treatment of the cutaneousmanifestations of erythema nodosum leprosum in leprosy. Physicians' DeskReference, 1154-1158 (56th ed., 2002). Because its administration topregnant women can cause birth defects, the sale of thalidomide isstrictly controlled. Id. Thalidomide has reportedly been studied in thetreatment of other diseases, such as chronic graft-vs-host disease,rheumatoid arthritis, sarcoidosis, several inflammatory skin diseases,and inflammatory bowel disease. See generally, Koch, H. P., Prog. Med.Chem. 22:165-242 (1985). See also, Moller, D. R., et al., J. Immunol.159:5157-5161 (1997); Vasiliauskas, E. A., et al., Gastroenterology117:1278-1287 (1999); Ehrenpreis, E. D., et al., Gastroenterology117:1271-1277 (1999). It has further been alleged that thalidomide canbe combined with other drugs to treat ischemia/repercussion associatedwith coronary and cerebral occlusion. See U.S. Pat. No. 5,643,915, whichis incorporated herein by reference.

More recently, thalidomide was found to exert immunomodulatory andanti-inflammatory effects in a variety of disease states, cachexia inAIDS, and opportunic infections in AIDS. In studies to define thephysiological targets of thalidomide, the drug was found to have a widevariety of biological activities exclusive of its sedative effectincluding neurotoxicity, teratogenicity, suppression of TNF-α productionby monocytes/macrophages and the accompanying inflammatory toxicitiesassociated with high levels of TNF-α, and inhibition of angiogenesis andneovascularization.

Additionally, beneficial effects have been observed in a variety ofdermatological conditions, ulcerative colitis, Crohn's disease,Bechets's syndrome, systemic lupus erythematosis, aphthous ulcers, andlupus. The anti-angiogenic properties of thalidomide in in vivo modelshave been reported. D'Amato et al., Thalidomide Is An Inhibitor OfAngiogenesis, 1994, PNAS, USA 91:4082-4085.

One of the most therapeutically significant potential uses ofthalidomide is in the treatment of cancer. The compound has beeninvestigated in the treatment of various types of cancer, such asrefractory multiple myeloma, brain, breast, colon, and prostate cancer,melanoma, mesothelioma, and renal cell carcinoma. See, e.g., Singhal,S., et al., New England J. Med. 341(21):1565-1571 (1999); and Marx, G.M., et al., Proc. Am. Soc. Clin. Oncology 18:454a (1999). Thalidomidereportedly can also be used to prevent the development of chroniccardiomyopathy in rats caused by doxorubicin. Costa, P. T., et al.,Blood 92(10: suppl. 1):235b (1998). Other reports concerning the use ofthalidomide in the treatment of specific cancers include its combinationwith carboplatin in the treatment of glioblastoma multiforme. McCann,J., Drug Topics 41-42 (Jun. 21, 1999). The use of thalidomide incombination with dexamethasone reportedly was effective in the treatmentof patients suffering from multiple myeloma who also received, assupportive care, human granulocyte colony-stimulating factor (G-CSF),ciprofloxacin, and non-absorbable antifungal agents. Kropff, M. H.,Blood 96(11 part 1):168a (2000); see also, Munshi, N. et al., Blood94(10 part 1):578a (1999). Other chemotherapy combinations that comprisethalidomide are disclosed in International Application No.PCT/US01/15326 to R. Govindarjan and A. Zeitlan, and in InternationalApplication No. PCT/US01/15327 to J. B. Zeldis, et al.

In an effort to provide compounds that have greater therapeutic safetyand efficacy than thalidomide, researchers have begun investigating alarge number of other compounds, some of which are derivatives ofthalidomide. See, e.g., Marriott, J. B., et al., Expert Opin. Biol.Ther. 1(4):1-8 (2001); G. W. Muller, et al., Journal of MedicinalChemistry 39(17): 3238-3240 (1996); and G. W. Muller, et al., Bioorganic& Medicinal Chemistry Letters 8: 2669-2674 (1998). Examples include, butare not limited to, the substituted 2-(2,6-dioxopiperidin-3-yl)phthalimies and substituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolesdescribed in U.S. Pat. Nos. 6,281,230 and 6,316,471, both to G. W.Muller, et al.

A group of compounds selected for their capacity to potently inhibitTNF-α production by LPS stimulated PBMC has been investigated. L. G.Corral, et al., Ann. Rheum. Dis. 58: (Suppl 1) 1107-1113 (1999). Thesecompounds, which are referred to as IMiDs™ or Immunomodulatory Drugs,show not only potent inhibition of TNF-α but also marked inhibition ofLPS induced monocyte IL1β and IL12 production. LPS induced IL6 is alsoinhibited by IMiDs™, albeit partially. These compounds are potentstimulators of LPS induced IL10, increasing IL10 levels by 200 to 300%.Id.

While many such compounds have shown promise as therapeutic agents,their mechanisms of action and effectiveness are still underinvestigation. Moreover, there remains a need for therapeutic agents totreat MDS and its related disorders.

3. SUMMARY OF THE INVENTION

This invention encompasses methods of treating or preventingmyelodysplastic syndrome (“MDS”) which comprise administering to apatient in need thereof a therapeutically or prophylactically effectiveamount of an immunomodulatory compound of the invention or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof. The invention also encompasses methods ofmanaging MDS (e.g., lengthening the time of remission) which compriseadministering to a patient in need of such management a therapeuticallyor prophylactically effective amount of an immunomodulatory compound ofthe invention, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof.

One embodiment of the invention encompasses the use of one or moreimmunomodulatory compounds in combination with conventional therapiespresently used to treat, prevent or manage MDS such as hematopoieticgrowth factors, cytokines, cancer chemotherapeutics, stem celltransplantation and other transplantations.

The invention further encompasses pharmaceutical compositions, singleunit dosage forms, and kits suitable for use in treating, preventingand/or managing MDS, which comprise an immunomodulatory compound of theinvention, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof.

4. DETAILED DESCRIPTION OF THE INVENTION

A first embodiment of the invention encompasses methods of treating orpreventing MDS which comprise administering to a patient in need of suchtreatment or prevention a therapeutically or prophylactically effectiveamount of an immunomodulatory compound, or a pharmaceutically acceptablesalt, solvate, hydrate, stereoisomer, clathrate, or prodrug thereof. Theembodiment encompasses the treatment, prevention or management ofspecific sub-types of MDS such as refractory anemia, refractory anemiawith ringed sideroblasts, refractory anemia with excess blasts,refractory anemia with excess blasts in transformation and chronicmyelomonocytic leukemia.

As used herein, the term “myelodysplastic syndromes” or “MDS” meanshematopoietic stem cell disorders characterized by one or more of thefollowing: ineffective blood cell production, progressive cytopenias,risk of progression to acute leukemia or cellular marrow with impairedmorphology and maturation (dysmyelopoiesis). The term “myelodysplasticsyndromes” or “MDS” unless otherwise noted includes: refractory anemia,refractory anemia with ringed sideroblasts, refractory anemia withexcess blasts, refractory anemia with excess blasts in transformationand chronic myelomonocytic leukemia.

Another embodiment of the invention encompasses methods of managing MDSwhich comprises administering to a patient in need of such management aprophylactically effective amount of an immunomodulatory compound, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof.

Another embodiment of the invention encompasses a pharmaceuticalcomposition comprising an immunomodulatory compound, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof.

Also encompassed by the invention are single unit dosage formscomprising an immunomodulatory compound, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof.

Another embodiment of the invention encompasses a kit comprising: apharmaceutical composition comprising an immunomodulatory compound, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof and a second active or dexamethasone orinstructions for use. The invention further encompasses kits comprisingsingle unit dosage forms.

One embodiment of the invention encompasses a method of treating,preventing and/or managing MDS, which comprises administering to apatient in need of such treatment, prevention and/or management atherapeutically or prophylactically effective amount of animmunomodulatory compound, or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, and atherapeutically or prophylactically effective amount of a second activeagent.

The second active agent is preferably a hematopoietic growth factor, acytokine, an anti-cancer agent, an antibiotic, an anti-fungal, ananti-inflammatory, an immunosuppressive agent such as a cyclosporin,conventional therapy for MDS, or other chemotherapeutic agent found forexample in the Physician's Desk Reference 2002. Preferred anti-cancer orcancer chemotherapeutics are apoptosis inducing agents, topoisomeraseinhibitors, anti-angiogenesis compounds, microtubule stabilizing agents,alkylating agents and other known conventional cancer chemotherapy. Mostpreferred second active agents are those capable of affecting orimproving blood production. Second active agents can be large molecules(e.g., proteins) or small molecules (e.g., synthetic inorganic,organometallic, or organic molecules). The examples of specific secondactive agent include, but are not limited to, etanercept (Enbrel®),imatinib (Glivec®), anti-TNF-α antibodies, infliximab (Remicade®),G-CSF, GM-CSF, EPO, topotecan, irinotecan, pentoxifylline,ciprofloxacin, dexamethasone, IL2, IL8, IL18, Ara-C, vinorelbine,vinblastine, isotretinoin, and 13-cis-retinoic acid. This invention alsoencompasses the use of native, naturally occurring, and recombinantproteins. The invention further encompasses mutants and derivatives(e.g., modified forms) of naturally occurring proteins that exhibit, invivo, at least some of the pharmacological activity of the proteins uponwhich they are based. Examples of mutants include, but are not limitedto, proteins that have one or more amino acid residues that differ fromthe corresponding residues in the naturally occurring forms of theproteins. Also encompassed by the term “mutants” are proteins that lackcarbohydrate moieties normally present in their naturally occurringforms (e.g., nonglycosylated forms). Examples of derivatives include,but are not limited to, pegylated derivatives and fusion proteins, suchas proteins formed by fusing IgG1 or IgG3 to the protein or activeportion of the protein of interest. See, e.g., Penichet, M. L. andMorrison, S. L., J. Immunol. Methods 248:91-101 (2001). Vaccines thatcause the secretion of proteins disclosed herein as well aspharmacologically active mutants, derivatives, and fusion thereof arealso encompassed by the invention.

Without being limited by theory, it is believed that certainimmunomodulatory compounds and proteins can act in complementary orsynergistic ways in the treatment or management of MDS. It is alsobelieved that certain proteins may reduce or eliminate particularadverse effects associated with some immunomodulatory compounds, therebyallowing the administration of larger amounts of an immunomodulatorycompound to patients and/or increasing patient compliance. It is furtherbelieved that some immunomodulatory compounds may reduce or eliminateparticular adverse effects associated with some protein-based MDStherapies, thereby allowing the administration of larger amounts ofprotein to patients and/or increasing patient compliance.

Another embodiment of the invention encompasses a method of reversing,reducing or avoiding an adverse effect associated with theadministration of a chemotherapeutics or therapeutics used to treatcancer or MDS in a patient suffering from MDS, which comprisesadministering to a patient in need thereof a therapeutically orprophylactically effective amount of an immunomodulatory compound, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof.

As inevitable leukemic transformation develops in certain stages of MDS,transplantation of peripheral blood stem cells, hematopoietic stem cellpreparation or bone marrow may be necessary. It is believed that thecombined use of an immunomodulatory compound and transplantation of stemcells in a patient suffering from MDS provides a unique and unexpectedsynergism. In particular, without being limited by theory, it isbelieved that an immunomodulatory compound exhibits immunomodulatoryactivity that may provide additive or synergistic effects when givenconcurrently with transplantation therapy. Immunomodulatory compoundscan work in combination with transplantation therapy reducingcomplications associated with the invasive procedure of transplantationand risk of related Graft Versus Host Disease (GVHD). Therefore, thisinvention encompasses a method of treating, preventing and/or managingMDS, which comprises administering to a patient (e.g., a human) animmunomodulatory compound, or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, before,during, or after transplantation therapy.

The invention also encompasses pharmaceutical compositions, single unitdosage forms, and kits which comprise one or more immunomodulatorycompounds, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof, a second active ingredient,and/or blood or cells for transplantation therapy. For example, the kitmay contain one or more compounds of the invention, stem cells fortransplantation and an immunosuppressive agent, antibiotic or otherdrug, each of which is to be used to treat the MDS patient.

4.1. Immunomodulatory Compounds

Compounds used in the invention include immunomodulatory compounds thatare racemic, stereomerically enriched or stereomerically pure, andpharmaceutically acceptable salts, solvates, hydrates, stereoisomers,clathrates, and prodrugs thereof. Preferred compounds used in theinvention are small organic molecules having a molecular weight lessthan about 1000 g/mol, and are not proteins, peptides, oligonucleotides,oligosaccharides or other macromolecules.

As used herein and unless otherwise indicated, the term “stereomericallypure” means a composition that comprises one stereoisomer of a compoundand is substantially free of other stereoisomers of that compound. Forexample, a stereomerically pure composition of a compound having onechiral center will be substantially free of the opposite enantiomer ofthe compound. A stereomerically pure composition of a compound havingtwo chiral centers will be substantially free of other diastereomers ofthe compound. A typical stereomerically pure compound comprises greaterthan about 80% by weight of one stereoisomer of the compound and lessthan about 20% by weight of other stereoisomers of the compound, morepreferably greater than about 90% by weight of one stereoisomer of thecompound and less than about 10% by weight of the other stereoisomers ofthe compound, even more preferably greater than about 95% by weight ofone stereoisomer of the compound and less than about 5% by weight of theother stereoisomers of the compound, and most preferably greater thanabout 97% by weight of one stereoisomer of the compound and less thanabout 3% by weight of the other stereoisomers of the compound. As usedherein and unless otherwise indicated, the term “stereomericallyenriched” means a composition that comprises greater than about 60% byweight of one stereoisomer of a compound, preferably greater than about70% by weight, more preferably greater than about 80% by weight of onestereoisomer of a compound. As used herein and unless otherwiseindicated, the term “enantiomerically pure” means a stereomerically purecomposition of a compound having one chiral center. Similarly, the term“stereomerically enriched” means a stereomerically enriched compositionof a compound having one chiral center.

As used herein and unless otherwise indicated, the term“immunomodulatory compounds” or “IMiDs™” (Celgene Corporation) usedherein encompasses small organic molecules that markedly inhibit TNF-α,LPS induced monocyte IL1β and IL12, and partially inhibit IL6production. Specific immunomodulatory compounds of the invention arediscussed below.

TNF-α is an inflammatory cytokine produced by macrophages and monocytesduring acute inflammation. TNF-α is responsible for a diverse range ofsignaling events within cells. TNF-α may play a pathological role incancer. Without being limited by particular theory, one of thebiological effects exerted by the immunomodulatory compounds of theinvention is the reduction of synthesis of TNF-α. Immunomodulatorycompounds of the invention enhance the degradation of TNF-α mRNA.

Further, without being limited by particular theory, immunomodulatorycompounds used in the invention may also be potent co-stimulators of Tcells and increase cell proliferation dramatically in a dose dependentmanner. Immunomodulatory compounds of the invention may also have agreater co-stimulatory effect on the CD8+ T cell subset than on the CD4+T cell subset. In addition, the compounds preferably haveanti-inflammatory properties, and efficiently co-stimulate T cells.

Specific examples of immunomodulatory compounds of the invention,include, but are not limited to, cyano and carboxy derivatives ofsubstituted styrenes such as those disclosed in U.S. Pat. No. 5,929,117;1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl) isoindolines and1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl) isoindolines such asthose described in U.S. Pat. No. 5,874,448; the tetra substituted2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolines described in U.S. Pat. No.5,798,368; 1-oxo and 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines(e.g., 4-methyl derivatives of thalidomide and EM-12), including, butnot limited to, those disclosed in U.S. Pat. No. 5,635,517; and a classof non-polypeptide cyclic amides disclosed in U.S. Pat. Nos. 5,698,579and 5,877,200; analogs and derivatives of thalidomide, includinghydrolysis products, metabolites, derivatives and precursors ofthalidomide, such as those described in U.S. Pat. Nos. 5,593,990,5,629,327, and 6,071,948 to D'Amato; aminothalidomide, as well asanalogs, hydrolysis products, metabolites, derivatives and precursors ofaminothalidomide, and substituted 2-(2,6-dioxopiperidin-3-yl)phthalimides and substituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolessuch as those described in U.S. Pat. Nos. 6,281,230 and 6,316,471;isoindole-imide compounds such as those described in U.S. patentapplication Ser. No. 09/972,487 filed on Oct. 5, 2001, U.S. patentapplication Ser. No. 10/032,286 filed on Dec. 21, 2001, andInternational Application No. PCT/US01/50401 (International PublicationNo. WO 02/059106). The entireties of each of the patents identifiedherein are incorporated herein by reference. Immunomodulatory compoundsof the invention do not include thalidomide.

Other specific immunomodulatory compounds of the invention include, butare not limited to, 1-oxo- and 1,3 dioxo-2-(2,6-dioxopiperidin-3-yl)isoindolines substituted with amino in the benzo ring as described inU.S. Pat. No. 5,635,517 which is incorporated herein. These compoundshave the structure I:

in which one of X and Y is C═O, the other of X and Y is C═O or CH₂, andR² is hydrogen or lower alkyl, in particular methyl. Specificimmunomodulatory compounds include, but are not limited to:

-   1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline;-   1-oxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline;-   1-oxo-2-(2,6-dioxopiperidin-3-yl)-6-aminoisoindoline;-   1-oxo-2-(2,6-dioxopiperidin-3-yl)-7-aminoisoindoline;-   1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline;-   and 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline.

Other specific immunomodulatory compounds of the invention belong to aclass of substituted 2-(2,6-dioxopiperidin-3-yl) phthalimides andsubstituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles, such as thosedescribed in U.S. Pat. Nos. 6,281,230; 6,316,471; 6,335,349; and6,476,052, and International Patent Application No. PCT/US97/13375(International Publication No. WO 98/03502), each of which isincorporated herein. Compounds representative of this class are of theformulas:

wherein R¹ is hydrogen or methyl. In a separate embodiment, theinvention encompasses the use of enantiomerically pure forms (e.g.optically pure (R) or (S) enantiomers) of these compounds.

Still other specific immunomodulatory compounds of the invention belongto a class of isoindole-imides disclosed in U.S. patent application Ser.Nos. 10/032,286 and 09/972,487, and International Application No.PCT/US01/50401 (International Publication No. WO 02/059106), each ofwhich are incorporated herein by reference. Representative compounds areof formula II:

and pharmaceutically acceptable salts, hydrates, solvates, clathrates,enantiomers, diastereomers, racemates, and mixtures of stereoisomersthereof, wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R¹ is H, (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, C(O)R³C(S)R³, C(O)OR⁴,(C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, C(O)NHR³,C(S)NHR³, C(O)NR³R^(3′), C(S)NR³R^(3′) or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H, F, benzyl, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or (C₂-C₈)alkynyl;

R³ and R^(3′) are independently (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl,(C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵;

R⁴ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkyl-OR⁵,benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, or(C₀-C₄)alkyl-(C₂-C₅)heteroaryl;

R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, or(C₂-C₅)heteroaryl;

each occurrence of R⁶ is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₂-C₅)heteroaryl, or(C₀-C₈)alkyl-C(O)O—R⁵ or the R⁶ groups can join to form aheterocycloalkyl group;

n is 0 or 1; and

* represents a chiral-carbon center.

In specific compounds of formula II, when n is 0 then R1 is(C3-C7)cycloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, benzyl, aryl,(C0-C4)alkyl-(C1-C6)heterocycloalkyl, (C0-C4)alkyl-(C2-C5)heteroaryl,C(O)R3, C(O)OR4, (C1-C8)alkyl-N(R6)2, (C1-C8)alkyl-OR⁵,(C1-C8)alkyl-C(O)OR5, C(S)NHR3, or (C1-C8)alkyl-O(CO)R5;

R² is H or (C₁-C₈)alkyl; and

R³ is (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,benzyl, aryl, (C₀-C₄)alkyl-C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-C₂-C₅)heteroaryl, (C₅-C₈)alkyl-N(R⁶)₂;(C₀-C₈)alkyl-NH—C(O)O—R⁵; (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵; and the other variables have the samedefinitions.

In other specific compounds of formula II, R² is H or (C₁-C₄)alkyl.

In other specific compounds of formula II, R¹ is (C₁-C₈)alkyl or benzyl.

In other specific compounds of formula II, R¹ is H(C₁-C₈)alkyl, benzyl,CH₂OCH₃, CH₂CH₂OCH₃, or

In another embodiment of the compounds of formula II, R¹ is

wherein Q is O or S, and each occurrence of R⁷ is independently H,(C₁-C₈)alkyl, benzyl, CH₂OCH₃, or CH₂CH₂OCH₃.

In other specific compounds of formula II, R¹ is C(O)R³.

In other specific compounds of formula II, R³ is(C₀-C₄)alkyl-C₂-C₅)heteroaryl, (C₁-C₈)alkyl, aryl, or (C₀-C₄)alkyl-OR⁵.

In other specific compounds of formula II, heteroaryl is pyridyl, furyl,or thienyl.

In other specific compounds of formula II, R¹ is C(O)OR⁴.

In other specific compounds of formula II, the H of C(O)NHC(O) can bereplaced with (C₁-C₄)alkyl, aryl, or benzyl.

Still other specific immunomodulatory compounds of the invention belongto a class of isoindole-imides disclosed in U.S. patent application Ser.No. 09/781,179, International Publication No. WO 98/54170, and U.S. Pat.No. 6,395,754, each of which are incorporated herein by reference.Representative compounds are of formula III:

and pharmaceutically acceptable salts, hydrates, solvates, clathrates,enantiomers, diastereomers, racemates, and mixtures of stereoisomersthereof, wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R is H or CH2OCOR′;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, or R⁴ is nitro or —NHR⁵ and the remaining of R¹, R²,R³, or R⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbons

R⁶ hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R′ is R⁷—CHR¹⁰—N(R⁸R⁹);

R⁷ is m-phenylene or p-phenylene or —(C_(n)H₂n)— in which n has a valueof 0 to 4;

each of R8 and R9 taken independently of the other is hydrogen or alkylof 1 to 8 carbon atoms, or R8 and R9 taken together are tetramethylene,pentamethylene, hexamethylene, or —CH₂CH₂[X]X₁CH₂CH₂— in which [X]X₁ is—O—, —S—, or —NH—;

R¹⁰ is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and

* represents a chiral-carbon center.

The most preferred immunomodulatory compounds of the invention are4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione and3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione. Thecompounds can be obtained via standard, synthetic methods (see e.g.,U.S. Pat. No. 5,635,517, incorporated herein by reference). Thecompounds are available from Celgene Corporation, Warren, N.J.4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (ACTIMID™)has the following chemical structure:

3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(REVIMID™) has the following chemical structure:

The compounds of the invention can either be commercially purchased orprepared according to the methods described in the patents or patentpublications disclosed herein. Further, optically pure compounds can beasymmetrically synthesized or resolved using known resolving agents orchiral columns as well as other standard synthetic organic chemistrytechniques.

As used herein and unless otherwise indicated, the term“pharmaceutically acceptable salt” encompasses non-toxic acid and baseaddition salts of the compound to which the term refers. Acceptablenon-toxic acid addition salts include those derived from organic andinorganic acids or bases know in the art, which include, for example,hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid,methanesulphonic acid, acetic acid, tartaric acid, lactic acid, succinicacid, citric acid, malic acid, maleic acid, sorbic acid, aconitic acid,salicylic acid, phthalic acid, embolic acid, enanthic acid, and thelike.

Compounds that are acidic in nature are capable of forming salts withvarious pharmaceutically acceptable bases. The bases that can be used toprepare pharmaceutically acceptable base addition salts of such acidiccompounds are those that form non-toxic base addition salts, i.e., saltscontaining pharmacologically acceptable cations such as, but not limitedto, alkali metal or alkaline earth metal salts and the calcium,magnesium, sodium or potassium salts in particular. Suitable organicbases include, but are not limited to, N,N-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumaine(N-methylglucamine), lysine, and procaine.

As used herein and unless otherwise indicated, the term “prodrug” meansa derivative of a compound that can hydrolyze, oxidize, or otherwisereact under biological conditions (in vitro or in vivo) to provide thecompound. Examples of prodrugs include, but are not limited to,derivatives of immunomodulatory compounds of the invention that comprisebiohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzableesters, biohydrolyzable carbamates, biohydrolyzable carbonates,biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Otherexamples of prodrugs include derivatives of immunomodulatory compoundsof the invention that comprise —NO, —NO₂, —ONO, or —ONO₂ moieties.Prodrugs can typically be prepared using well-known methods, such asthose described in 1 Burger's Medicinal Chemistry and Drug Discovery,172-178, 949-982 (Manfred E. Wolff ed., 5th ed. 1995), and Design ofProdrugs (H. Bundgaard ed., Elselvier, New York 1985).

As used herein and unless otherwise indicated, the terms“biohydrolyzable amide,” “biohydrolyzable ester,” “biohydrolyzablecarbamate,” “biohydrolyzable carbonate,” “biohydrolyzable ureide,”“biohydrolyzable phosphate” mean an amide, ester, carbamate, carbonate,ureide, or phosphate, respectively, of a compound that either: 1) doesnot interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable esters include, but are not limited to,lower alkyl esters, lower acyloxyalkyl esters (such as acetoxylmethyl,acetoxyethyl, aminocarbonyloxymethyl, pivaloyloxymethyl, andpivaloyloxyethyl esters), lactonyl esters (such as phthalidyl andthiophthalidyl esters), lower alkoxyacyloxyalkyl esters (such asmethoxycarbonyloxymethyl, ethoxycarbonyloxyethyl andisopropoxycarbonyloxyethyl esters), alkoxyalkyl esters, choline esters,and acylamino alkyl esters (such as acetamidomethyl esters). Examples ofbiohydrolyzable amides include, but are not limited to, lower alkylamides, α-amino acid amides, alkoxyacyl amides, andalkylaminoalkylcarbonyl amides. Examples of biohydrolyzable carbamatesinclude, but are not limited to, lower alkylamines, substitutedethylenediamines, aminoacids, hydroxyalkylamines, heterocyclic andheteroaromatic amines, and polyether amines.

It should be noted that if there is a discrepancy between a depictedstructure and a name given that structure, the depicted structure is tobe accorded more weight. In addition, if the stereochemistry of astructure or a portion of a structure is not indicated with, forexample, bold or dashed lines, the structure or portion of the structureis to be interpreted as encompassing all stereoisomers of it.

4.2. Second Active Agents

One or more second active ingredients can be used in the methods andcompositions of the invention together with an immunomodulatory compoundof the invention. In a preferred embodiment, the second active agentsare capable of affecting or improving the process of blood cellproduction. Specific second active agents also stimulate the divisionand differentiation of committed erythroid progenitors in cells in vitroor in vivo.

Second active agents can be large molecules (e.g., proteins) or smallmolecules (e.g., synthetic inorganic, organometallic, or organicmolecules). The second active agents include but are not limited tohematopoietic growth factors, cytokines, anti-cancer agents,antibiotics, proteasome inhibitors, immunosuppressive agents and othertherapeutics discussed herein. Particular agents include, but are notlimited to, G-CSF, GM-CSF, EPO, dexamethasone, topotecan,pentoxifylline, irinotecan, ciprofloxacin, vinorelbine, IL2, IL8, IL18,Ara-C, isotretinoin, 13-cis-retinoic acid,12-O-tetradecanoylphorbol-13-acetate (TPA), 5-AZA2′-deoyxcytidine,9-nitrocamp-tothecin, transretinoic acid, amifostine, amphotericin B andliposomal amphotericin B, anti-CD-20 monoclonal antibody, anti-thymocyleglobulin (ATG), arsenic trioxide, azacytidine, bevacizumab, bismuthmonoclonal antibody, bryostatin, busulfan, caspofungin acetate,celocoxib, cladribine, cyclophosphamide, cyclosporine, cytarabine,cytosine, daunorubicin, depsipeptide, etoposide, farresy transferaseinhibitor, flavopiridol, Flt3 ligand, fludarabine, gentuzumab ozogomicin(mylotarg), etanercept (Enbrel®), imatinib (Glivec®), anti-TNF-αantibodies, infliximab (Remicade®), humanized monoclonal anti-VEGFantibody, idarubicine, leucovorin, melphalan, mitoxantrone, monoclonalantibody ABX-CBL, monoclonal antibody CD52, mycophenolate mofetil,oblimersen, omega-3 fatty acids, pentostatin, phenylbutyrate, PR1leukemia peptide vaccine, montanide, proteasome inhibitor, sodiumphenyl-butyrate, sodium salicylate, temozolomide, thymoglobulin,troxatyl, tumor necrosis factor receptor IgG chimera, Yttrium Y 90humanized monoclonal antibody M195. In a specific embodiment of theinvention, an immunomodulatory compound of the invention is used incombination with pentoxifylline, ciprofloxacin, and/or dexamethasone.

This invention also encompasses the use of native, naturally occurring,and recombinant proteins. The invention further encompasses mutants andderivatives (e.g., modified forms) of naturally occurring proteins thatexhibit, in vivo, at least some of the pharmacological activity of theproteins upon which they are based. Examples of mutants include, but arenot limited to, proteins that have one or more amino acid residues thatdiffer from the corresponding residues in the naturally occurring formsof the proteins. Also encompassed by the term “mutants” are proteinsthat lack carbohydrate moieties normally present in their naturallyoccurring forms (e.g., nonglycosylated forms). Examples of derivativesinclude, but are not limited to, pegylated derivatives and fusionproteins, such as proteins formed by fusing IgG1 or IgG3 to the proteinor active portion of the protein of interest. See, e.g., Penichet, M. L.and Morrison, S. L., J. Immunol. Methods 248:91-101 (2001).

Recombinant and mutated forms of G-CSF can be prepared as described inU.S. Pat. Nos. 4,810,643; 4,999,291; 5,528,823; and 5,580,755; all ofwhich are incorporated herein by reference. Recombinant and mutatedforms of GM-CSF can be prepared as described in U.S. Pat. Nos.5,391,485; 5,393,870; and 5,229,496; all of which are incorporatedherein by reference. In fact, recombinant forms of G-CSF and GM-CSF arecurrently sold in the United States for the treatment of symptomsassociated with specific chemotherapies. A recombinant form of G-CSFknown as filgrastim is sold in the United States under the trade nameNEUPOGEN®. NEUPOGEN® is known to stimulate division and maturation ofgranulocytes, mostly neutrophils, in MDS patients and to enhanceerythroid response in combination with EPO. Physicians' Desk Reference,587-592 (56^(th) ed., 2002). A recombinant form of GM-CSF known assargramostim is also sold in the United States under the trade nameLEUKINE®. LEUKINE® is known to stimulate division and maturation ofearlier myeloid and macrophage precursor cells and has been reported toincrease granulocytes. Physicians' Desk Reference, 1755-1760 (56^(th)ed., 2002). A recombinant form of EPO known as epoetin alfa is sold inthe United States under the trade name EPOGEN®. EPOGEN® is used tostimulate red cell production by stimulating division and maturation ofcommitted red cell precursor cells. EPOGEN® has been reported to beeffective in 20-26% of MDS patient when administered by itself and in asmany as 48% of patients when combined with G-CSF or GM-CSF. Physicians'Desk Reference, 582-587 (56^(th) ed., 2002).

A growth-factor or cytokine such as G-CSF, GM-CSF and EPO can also beadministered in the form of a vaccine. For example, vaccines thatsecrete, or cause the secretion of, cytokines such as G-CSF and GM-CSFcan be used in the methods, pharmaceutical compositions, and kits of theinvention. See, e.g., Emens, L. A., et al., Curr. Opinion Mol. Ther.3(1):77-84 (2001).

Other compounds that can be administered or used in combination with animmunomodulatory compound of the invention include those disclosed inU.S. provisional patent application No. 60/380,842, filed May 17, 2002,and U.S. provisional patent application No. 60/380,843, filed May 17,2002, both of which are incorporated herein by reference.

4.3. Methods of Treatment and Management

Methods of this invention encompass methods of preventing, treatingand/or managing various types of MDS. As used herein, unless otherwisespecified, the term “preventing” includes but is not limited to,inhibition or the averting of symptoms associated with MDS. The symptomsassociated with MDS include, but are not limited to, anemia,thrombocytopenia, neutropenia, cytopenia, bicytopenia (two deficientcell lines), and pancytopenia (three deficient cell lines). As usedherein, unless otherwise specified, the term “treating” refers to theadministration of a composition after the onset of symptoms of MDS,whereas “preventing” refers to the administration prior to the onset ofsymptoms, particularly to patients at risk of MDS. As used herein andunless otherwise indicated, the term “managing” encompasses preventingthe recurrence of MDS in a patient who had suffered from MDS,lengthening the time a patient who had suffered from MDS remains inremission, and/or preventing the occurrence of MDS in patients at riskof suffering from MDS.

The invention encompasses methods of treating or preventing patientswith primary and secondary MDS. It further encompasses methods treatingpatients who have been previously treated for MDS, as well as those whohave not previously been treated for MDS. Because patients with MDS haveheterogenous clinical manifestations and varying clinical outcomes, ithas become apparent that staging the patients according to theirprognosis and approaching therapy depending on the severity and stage isnecessary. Indeed, the methods and compositions of this invention can beused in various stages of treatments for patients with one or more typesof MDS including, but not limited to, refractory anemia (RA), RA withringed sideroblasts (RARS), RA with excess blasts (RAEB), RAEB intransformation (RAEB-T), or chronic myelomonocytic leukemia (CMML). Theinvention also contemplates treating patients diagnosed using the IPSSfor MDS discussed above. Greenberg et al., Blood 1997 (89):2079-88.

Methods encompassed by this invention comprise administering animmunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof to a patient (e.g., a human) suffering, or likely to suffer,from MDS. Specific patient populations include the elderly, i.e., ages60 and above as well as those over 35 years of age. Patients withfamilial history of MDS or leukemia are also preferred candidates forpreventive regimens.

In one embodiment of the invention, an immunomodulatory compound of theinvention is administered orally and in a single or divided daily dosesin an amount of from about 0.10 to about 150 mg/day. In a particularembodiment, 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione(Actimid™) is administered in an amount of from about 0.1 to about 1 mgper day, or alternatively about 5 mg every other day.3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(Revimid™) can be preferably administered in an amount of from about 5to 25 mg per day, or alternatively from about 25 to about 50 mg everyother day.

4.3.1 Combination Therapy with a Second Active Agent

Particular methods of the invention comprise comprises administering 1)an immunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof, and 2) a second active agent or active ingredient. Examples ofimmunomodulatory compounds of the invention are disclosed herein (see,e.g., section 4.1); and examples of the second active agents are alsodisclosed herein (see, e.g., section 4.2).

Administration of the immunomodulatory compounds and the second activeagents to a patient can occur simultaneously or sequentially by the sameor different routes of administration. The suitability of a particularroute of administration employed for a particular active agent willdepend on the active agent itself (e.g., whether it can be administeredorally without decomposing prior to entering the blood stream) and thedisease being treated. A preferred route of administration for animmunomodulatory compound is oral. Preferred routes of administrationfor the second active agents or ingredients of the invention are knownto those of ordinary skill in the art. See, e.g., Physicians' DeskReference, 1755-1760 (56^(th) ed., 2002).

In one embodiment, the second active agent is administered intravenouslyor subcutaneously and once or twice daily in an amount of from about 1to about 1000 mg, from about 5 to about 500 mg, from about 10 to about350 mg, or from about 50 to about 200 mg. The specific amount of thesecond active agent will depend on the specific agent used, the type ofMDS being treated or managed, the severity and stage of MDS, and theamount(s) of immunomodulatory compounds of the invention and anyoptional additional active agents concurrently administered to thepatient. In a particular embodiment, the second active agent is GM-CSF,G-CSF, EPO, transretinoic acid, dexamethasone, topotecan,pentoxifylline, ciprofloxacin, dexamethasone, IL2, IL8, IL18, Ara-C,vinorelbine, or a combination thereof. GM-CSF is administered in anamount of from about 60 to about 500 mcg/m² intravenously over 2 hours,or from about 5 to about 12 mcg/m²/day subcutaneously. G-CSF isadministered subcutaneously in an amount of about 1 mcg/kg/day initiallyand can be adjusted depending on rise of total granulocyte counts. Themaintenance dose is 300 (in smaller patients) or 480 mcg subcutaneously.EPO is administered subcutaneously in an amount of 10,000 Unit 3 timesper week.

4.3.2 Use with Transplantation Therapy

In still another embodiment, this invention encompasses a method oftreating, preventing and/or managing MDS, which comprises administeringthe immunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof, in conjunction with transplantation therapy. As discussedelsewhere herein, the treatment of MDS is based on the stages andmechanism of the disease. As inevitable leukemic transformation developsin certain stages of MDS, transplantation of peripheral blood stemcells, hematopoietic stem cell preparation or bone marrow may benecessary. The combined use of the immunomodulatory compound of theinvention and transplantation therapy provides a unique and unexpectedsynergism. In particular, an immunomodulatory compound of the inventionexhibits immunomodulatory activity that may provide additive orsynergistic effects when given concurrently with transplantation therapyin patients with MDS. An immunomodulatory compound of the invention canwork in combination with transplantation therapy reducing complicationsassociated with the invasive procedure of transplantation and risk ofrelated Graft Versus Host Disease (GVHD). This invention encompasses amethod of treating, preventing and/or managing MDS which comprisesadministering to a patient (e.g., a human) an immunomodulatory compoundof the invention, or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, clathrate, or prodrug thereof, before, during, orafter the transplantation of umbilical cord blood, placental blood,peripheral blood stem cell, hematopoietic stem cell preparation or bonemarrow. Examples of stem cells suitable for use in the methods of theinvention are disclosed in U.S. provisional patent application No.60/372,348, filed Apr. 12, 2002 by R. Hariri et al., the entirety ofwhich is incorporated herein by reference.

4.3.3. Cycling Therapy

In certain embodiments, the prophylactic or therapeutic agents of theinvention are cyclically administered to a patient. Cycling therapyinvolves the administration of a first agent for a period of time,followed by the administration of the agent and/or the second agent fora period of time and repeating this sequential administration. Cyclingtherapy can reduce the development of resistance to one or more of thetherapies, avoid or reduce the side effects of one of the therapies,and/or improves the efficacy of the treatment.

In a particular embodiment, prophylactic or therapeutic agents areadministered in a cycle of about 16 weeks, about once or twice everyday. One cycle can comprise the administration of a therapeutic orprophylactic agent and at least one (1) or three (3) weeks of rest. Thenumber of cycles administered is from about 1 to about 12 cycles, moretypically from about 2 to about 10 cycles, and more typically from about2 to about 8 cycles.

4.4. Pharmaceutical Compositions and Single Unit Dosage Forms

Pharmaceutical compositions can be used in the preparation ofindividual, single unit dosage forms. Pharmaceutical compositions anddosage forms of the invention comprise an immunomodulatory compound ofthe invention, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof. Pharmaceutical compositionsand dosage forms of the invention can further comprise one or moreexcipients.

Pharmaceutical compositions and dosage forms of the invention can alsocomprise one or more additional active ingredients. Consequently,pharmaceutical compositions and dosage forms of the invention comprisethe active ingredients disclosed herein (e.g., an immunomodulatorycompound of the invention, or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, and asecond active ingredient). Examples of optional additional activeingredients are disclosed herein (see, e.g., section 4.2).

Single unit dosage forms of the invention are suitable for oral, mucosal(e.g., nasal, sublingual, vaginal, buccal, or rectal), or parenteral(e.g., subcutaneous, intravenous, bolus injection, intramuscular, orintraarterial), transdermal or transcutaneous administration to apatent. Examples of dosage forms include, but are not limited to:tablets; caplets; capsules, such as soft elastic gelatin capsules;cachets; troches; lozenges; dispersions; suppositories; powders;aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage formssuitable for oral or mucosal administration to a patient, includingsuspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a patient.

The composition, shape, and type of dosage forms of the invention willtypically vary depending on their use. For example, a dosage form usedin the acute treatment of a disease may contain larger amounts of one ormore of the active ingredients it comprises than a dosage form used inthe chronic treatment of the same disease. Similarly, a parenteraldosage form may contain smaller amounts of one or more of the activeingredients it comprises than an oral dosage form used to treat the samedisease. These and other ways in which specific dosage forms encompassedby this invention will vary from one another will be readily apparent tothose skilled in the art. See, e.g., Remington's PharmaceuticalSciences, 18th ed., Mack Publishing, Easton Pa. (1990).

Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well known to those skilled inthe art of pharmacy, and non-limiting examples of suitable excipientsare provided herein. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a patient.For example, oral dosage forms such as tablets may contain excipientsnot suited for use in parenteral dosage forms. The suitability of aparticular excipient may also depend on the specific active ingredientsin the dosage form. For example, the decomposition of some activeingredients may be accelerated by some excipients such as lactose, orwhen exposed to water. Active ingredients that comprise primary orsecondary amines are particularly susceptible to such accelerateddecomposition. Consequently, this invention encompasses pharmaceuticalcompositions and dosage forms that contain little, if any, lactose othermono- or di-saccharides. As used herein, the term “lactose-free” meansthat the amount of lactose present, if any, is insufficient tosubstantially increase the degradation rate of an active ingredient.

Lactose-free compositions of the invention can comprise excipients thatare well known in the art and are listed, for example, in the U.S.Pharmacopeia (USP) 25-NF20 (2002). In general, lactose-free compositionscomprise active ingredients, a binder/filler, and a lubricant inpharmaceutically compatible and pharmaceutically acceptable amounts.Preferred lactose-free dosage forms comprise active ingredients,microcrystalline cellulose, pre-gelatinized starch, and magnesiumstearate.

This invention further encompasses anhydrous pharmaceutical compositionsand dosage forms comprising active ingredients, since water canfacilitate the degradation of some compounds. For example, the additionof water (e.g., 5%) is widely accepted in the pharmaceutical arts as ameans of simulating long-term storage in order to determinecharacteristics such as shelf-life or the stability of formulations overtime. See, e.g., Jens T. Carstensen, Drug Stability: Principles &Practice, 2d. Ed., Marcel Dekker, NY, N.Y., 1995, pp. 379-80. In effect,water and heat accelerate the decomposition of some compounds. Thus, theeffect of water on a formulation can be of great significance sincemoisture and/or humidity are commonly encountered during manufacture,handling, packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms of the inventioncan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine are preferablyanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions are preferably packaged using materials known to preventexposure to water such that they can be included in suitable formularykits. Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs, and strip packs.

The invention further encompasses pharmaceutical compositions and dosageforms that comprise one or more compounds that reduce the rate by whichan active ingredient will decompose. Such compounds, which are referredto herein as “stabilizers,” include, but are not limited to,antioxidants such as ascorbic acid, pH buffers, or salt buffers.

Like the amounts and types of excipients, the amounts and specific typesof active ingredients in a dosage form may differ depending on factorssuch as, but not limited to, the route by which it is to be administeredto patients. However, typical dosage forms of the invention comprise animmunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof in an amount of from about 0.10 to about 150 mg. Typical dosageforms comprise an immunomodulatory compound of the invention, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof in an amount of about 0.1, 1, 2, 5, 7.5,10, 12.5, 15, 17.5, 20, 25, 50, 100, 150 or 200 mg. In a particularembodiment, a preferred dosage form comprises4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (Actimid™) inan amount of about 1, 2, 5, 10, 25 or 50 mg. In a specific embodiment, apreferred dosage form comprises3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(Revimid™) in an amount of about 5, 10, 25 or 50 mg. Typical dosageforms comprise the second active ingredient in an amount of 1 to about1000 mg, from about 5 to about 500 mg, from about 10 to about 350 mg, orfrom about 50 to about 200 mg. Of course, the specific amount of thesecond active ingredient will depend on the specific agent used, thetype of MDS being treated or managed, and the amount(s) ofimmunomodulatory compounds of the invention, and any optional additionalactive agents concurrently administered to the patient.

4.4.1 Oral Dosage Forms

Pharmaceutical compositions of the invention that are suitable for oraladministration can be presented as discrete dosage forms, such as, butare not limited to, tablets (e.g., chewable tablets), caplets, capsules,and liquids (e.g., flavored syrups). Such dosage forms containpredetermined amounts of active ingredients, and may be prepared bymethods of pharmacy well known to those skilled in the art. Seegenerally, Remington's Pharmaceutical Sciences, 18th ed., MackPublishing, Easton Pa. (1990).

Typical oral dosage forms of the invention are prepared by combining theactive ingredients in an intimate admixture with at least one excipientaccording to conventional pharmaceutical compounding techniques.Excipients can take a wide variety of forms depending on the form ofpreparation desired for administration. For example, excipients suitablefor use in oral liquid or aerosol dosage forms include, but are notlimited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars,micro-crystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms, in which case solidexcipients are employed. If desired, tablets can be coated by standardaqueous or nonaqueous techniques. Such dosage forms can be prepared byany of the methods of pharmacy. In general, pharmaceutical compositionsand dosage forms are prepared by uniformly and intimately admixing theactive ingredients with liquid carriers, finely divided solid carriers,or both, and then shaping the product into the desired presentation ifnecessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free-flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms of theinvention include, but are not limited to, binders, fillers,disintegrants, and lubricants. Binders suitable for use inpharmaceutical compositions and dosage forms include, but are notlimited to, corn starch, potato starch, or other starches, gelatin,natural and synthetic gums such as acacia, sodium alginate, alginicacid, other alginates, powdered tragacanth, guar gum, cellulose and itsderivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropylmethyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystallinecellulose, and mixtures thereof.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICELRC-581, AVICEL-PH-105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. Anspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL-PH-103™ and Starch 1500LM.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions of the invention istypically present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

Disintegrants are used in the compositions of the invention to providetablets that disintegrate when exposed to an aqueous environment.Tablets that contain too much disintegrant may disintegrate in storage,while those that contain too little may not disintegrate at a desiredrate or under the desired conditions. Thus, a sufficient amount ofdisintegrant that is neither too much nor too little to detrimentallyalter the release of the active ingredients should be used to form solidoral dosage forms of the invention. The amount of disintegrant usedvaries based upon the type of formulation, and is readily discernible tothose of ordinary skill in the art. Typical pharmaceutical compositionscomprise from about 0.5 to about 15 weight percent of disintegrant,preferably from about 1 to about 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, agar-agar,alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate, potato or tapioca starch, other starches, pre-gelatinizedstarch, other starches, clays, other algins, other celluloses, gums, andmixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, andmixtures thereof. Additional lubricants include, for example, a syloidsilica gel (AEROSIL200, manufactured by W.R. Grace Co. of Baltimore,Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co.of Plano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants are typically used in an amount of less than about 1 weightpercent of the pharmaceutical compositions or dosage forms into whichthey are incorporated.

A preferred solid oral dosage form of the invention comprises animmunomodulatory compound of the invention, anhydrous lactose,microcrystalline cellulose, polyvinylpyrrolidone, stearic acid,colloidal anhydrous silica, and gelatin.

4.4.2 Delayed Release Dosage Forms

Active ingredients of the invention can be administered by controlledrelease means or by delivery devices that are well known to those ofordinary skill in the art. Examples include, but are not limited to,those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548,5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of which isincorporated herein by reference. Such dosage forms can be used toprovide slow or controlled-release of one or more active ingredientsusing, for example, hydropropylmethyl cellulose, other polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, microspheres, or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled-release formulations known to those of ordinary skill in theart, including those described herein, can be readily selected for usewith the active ingredients of the invention. The invention thusencompasses single unit dosage forms suitable for oral administrationsuch as, but not limited to, tablets, capsules, gelcaps, and capletsthat are adapted for controlled-release.

All controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood levels of the drug,and can thus affect the occurrence of side (e.g., adverse) effects.

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled-release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds.

4.4.3 Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients by variousroutes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. Becausetheir administration typically bypasses patients' natural defensesagainst contaminants, parenteral dosage forms are preferably sterile orcapable of being sterilized prior to administration to a patient.Examples of parenteral dosage forms include, but are not limited to,solutions ready for injection, dry products ready to be dissolved orsuspended in a pharmaceutically acceptable vehicle for injection,suspensions ready for injection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms ofthe invention are well known to those skilled in the art. Examplesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms of the invention. For example, cyclodextrin andits derivatives can be used to increase the solubility of animmunomodulatory compound of the invention, and its derivatives. See,e.g., U.S. Pat. No. 5,134,127, which is incorporated herein byreference.

4.4.4 Topical and Mucosal Dosage Forms

Topical and mucosal dosage forms of the invention include, but are notlimited to, sprays, aerosols, solutions, emulsions, suspensions, orother forms known to one of skill in the art. See, e.g., Remington'sPharmaceutical Sciences, 16^(th) and 18^(th) eds., Mack Publishing,Easton Pa. (1980 & 1990); and Introduction to Pharmaceutical DosageForms, 4th ed., Lea & Febiger, Philadelphia (1985). Dosage formssuitable for treating mucosal tissues within the oral cavity can beformulated as mouthwashes or as oral gels.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide topical and mucosal dosage forms encompassedby this invention are well known to those skilled in the pharmaceuticalarts, and depend on the particular tissue to which a givenpharmaceutical composition or dosage form will be applied. With thatfact in mind, typical excipients include, but are not limited to, water,acetone, ethanol, ethylene glycol, propylene glycol, butane-1,3-diol,isopropyl myristate, isopropyl palmitate, mineral oil, and mixturesthereof to form solutions, emulsions or gels, which are non-toxic andpharmaceutically acceptable. Moisturizers or humectants can also beadded to pharmaceutical compositions and dosage forms if desired.Examples of such additional ingredients are well known in the art. See,e.g., Remington's Pharmaceutical Sciences, 16^(th) and 18^(th) eds.,Mack Publishing, Easton Pa. (1980 & 1990).

The pH of a pharmaceutical composition or dosage form may also beadjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

4.4.5 Kits

Typically, active ingredients of the invention are preferably notadministered to a patient at the same time or by the same route ofadministration. This invention therefore encompasses kits which, whenused by the medical practitioner, can simplify the administration ofappropriate amounts of active ingredients to a patient.

A typical kit of the invention comprises a dosage form of animmunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, prodrug, or clathratethereof. Kits encompassed by this invention can further compriseadditional active ingredients such as G-CSF, GM-CSF, EPO, topotecan,pentoxifylline, ciprofloxacin, dexamethasone, IL2, IL8, IL18, Ara-C,vinorelbine, isotretinoin, 13-cis-retinoic acid, or a pharmacologicallyactive mutant or derivative thereof, or a combination thereof. Examplesof the additional active ingredients include, but are not limited to,those disclosed herein (see, e.g., section 4.2).

Kits of the invention can further comprise devices that are used toadminister the active ingredients. Examples of such devices include, butare not limited to, syringes, drip bags, patches, and inhalers.

Kits of the invention can further comprise cells or blood fortransplantation as well as pharmaceutically acceptable vehicles that canbe used to administer one or more active ingredients. For example, if anactive ingredient is provided in a solid form that must be reconstitutedfor parenteral administration, the kit can comprise a sealed containerof a suitable vehicle in which the active ingredient can be dissolved toform a particulate-free sterile solution that is suitable for parenteraladministration. Examples of pharmaceutically acceptable vehiclesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

5. EXAMPLES

The following studies are intended to further illustrate the inventionwithout limiting its scope.

Excessive production of the growth inhibitory cytokine TNF-α isdemonstrated in bone marrow plasma of patients with MDS, implicatingTNF-α as a critical negative regulator of erythroid progenitor survivalin the disorder. As a result, a study with an immunomodulatory compoundof the invention was conducted.

5.1. Pharmacology and Toxicology Studies

A series of non-clinical pharmacology and toxicology studies have beenperformed to support the clinical evaluation of an immunomodulatorycompound of the invention in human subjects. These studies wereperformed in accordance with internationally recognized guidelines forstudy design and in compliance with the requirements of Good LaboratoryPractice (GLP), unless otherwise noted.

The pharmacological properties of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione,including activity comparisons with thalidomide, have been characterizedin in vitro studies. Studies examined the effects of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione orthalidomide on the production of various cytokines. In all studies,3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione was atleast 50 times more potent than thalidomide. In addition, a safetypharmacology study of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione hasbeen conducted in dogs and the effects of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione on ECGparameters were examined further as part of three repeat-dose toxicitystudies in primates. The results of these studies are described below.

5.2. Modulation of Cytokine Production

Inhibition of TNF-α production following LPS-stimulation of human PBMCand human whole blood by3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione orthalidomide was investigated in vitro (Muller et al., Bioorg. Med. Chem.Lett. 9:1625-1630, 1999). The IC₅₀'s of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione forinhibiting production of TNF-α following LPS-stimulation of PBMC andhuman whole blood were ˜100 nM (25.9 ng/mL) and ˜480 nM (103.6 ng/mL),respectively. Thalidomide, in contrast, had an IC₅₀ of ˜194 μM (50.2μg/mL) for inhibiting production of TNF-α following LPS-stimulation ofPBMC.

In vitro studies suggest a pharmacological activity profile for3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione that issimilar to, but 50 to 2000 times more potent than, thalidomide. Thepharmacological effects of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione derivefrom its action as an inhibitor of cellular response toreceptor-initiated trophic signals (e.g., IGF-1, VEGF,cyclooxygenase-2), and other activities. As a result,3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionesuppresses the generation of inflammatory cytokines, down-regulatesadhesion molecules and apoptosis inhibitory proteins (e.g., cFLIP,cIAP), promotes sensitivity to death-receptor initiated programmed celldeath, and suppresses angiogenic response. The studies show that3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dioneabrogates mitogenic response to VEGF in AML cells by extinguishingligant-induced Akt-phosphorylation, and selectively suppresses MDS vsnormal bone marrow progenitor formation in pre-clinical models.

5.3. Clinical Studies in MDS Patients

Protocol

An immunomodulatory compound of the invention, such as4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione and3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione, isadministered in an amount of from about 0.1 to about 25 mg per day topatients with MDS for 16 weeks, who are subsequently evaluated for ahematological response. Response rates are assessed in cohortsstratified by the likelihood of an MDS subtype to transform to leukemiaaccording to the International Prognostic Scoring System (IPSS)-definedrisk groups (i.e., IPSS Low and Intermediate I; versus IPSS IntermediateII and High).

For example, fifteen patients are enrolled in the first cohort andreceive treatment with 25 mg per day of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione. Thenumber of patients who subsequently experience an erythroid response(major or minor response) by week 16 is evaluated. If no responses areobserved, the study is terminated due to lack of efficacy. If, however,4 or more patients respond, the study is terminated due to promisingclinical activity. In the intermediate case (e.g., 1, 2 or 3 patientsrespond), a second cohort of 10 patients is enrolled. If after thecompletion of treatment by the second cohort, 4 or more patients respondamong the 25 patients treated, it is concluded that the3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione showspromising clinical activity.

Clinical Study

Clinical studies were performed for the remitting potential of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione in MDSpatients with red blood cell transfusion-dependence (>4 units/8 weeks)or symptomatic anemia (Hgb<10 g/dl). Patients received continuoustreatment with3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione at aoral dose of 25 mg daily. Responses were assessed according to IWGcriteria after 16 weeks of treatments. Among 15 patients receiving thetreatments, 11 patients were evaluable for toxicity, nine patients wereevaluable for response (>8 wks therapy), and three patients discontinuedthe therapy prematurely (<2 weeks) due to cholecystitis, autoimmunehemolytic anemia, or patient refusal. Median age of the patients was 78years ranging from 51 to 82 years. FAB types of the MDS patients includeRA [4 patients], RARS [4 patients], RAEB [6 patients], and RAEB-T [1patient] with corresponding IPSS categories of Low/Int-1 in 11 patientsand Int-2/High in four patients. Myelosuppression, which wascharacterized by higher than grade 3 common toxicity criteria or 50%decrease in leukocyte and platelet counts [9 patients], and grade 3fatigue [1 patient], necessitated dose reduction to 10 mg in the initialten patients. All subsequent patients initiated oral administrationswith 10 mg daily. Grade 1,2 drug-related adverse effects were limited tothe 25 mg dose and included pruritus or itchy scalp [6 patients] andmyalgia [1 patient]. Six (66%) of nine evaluable patients experiencedhematologic benefit (dual lineage, 1 patient), including 6/7 (86%)patients with IPSS Low/Int-1. Hematologic responses included RBCtransfusion-independence [4 patients], decrease in RBC transfusions ofmore than 50% [1 patient], increase in Hgb of more than 1.5 g [1patient], and one minor platelet response (increase of more than30,000/μL). Among five patients evaluable for cytogenetic response,three patients achieved either a complete or partial (decrease inabnormal metaphases of more than 50%) remission. Responses wereassociated with normalization of blast percentage [1 patient], reducedgrade of BM cytologic dysplasia, and 50% to more than 40 timesimprovement in BM multipotent progenitor (CFU-GEMM) and erythroid burst(BFU-E) formation. Correlation with changes in apoptotic index,angiogenic features (cellular/plasma VEGF, microvessel density),cytokine generation, and proliferative fraction (Ki67) are in progress.The results of this study indicate that3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione hasremarkable erythropoietic and cytogenetic remitting activity in patientswith low/intermediate-1 risk MDS. Clinical benefit appears greatest inpatients with low/intermediate-1 disease or the 5q-syndrome, associatedwith resolution of cytology dysplasia. The increase in apoptotic index,restoration of CFC, and suppression of karyotypic abnormalities suggestthat the compound accelerates extinction of myelodysplastic clones.Based upon these data, the study has been expanded to treat additionalsubjects. Treatment with 10 mg as a continuous oral daily dose iswell-tolerated with minimal myelosuppression.

Expanded Study

The clinical study was expanded with additional 16 MDS patients for atleast eight weeks. According to the IPSS, 13 of these patients werecategorized as low- or intermediate-1-risk patients and three patientswere grouped as intermediate-2- or high-risk patients. According to theFAB classification, there were 11 patients with refractory anemia (RA)or RA with ringed sideroblasts (RARS), and five patients with RA withexcess blasts (RAEB), RAEB in transformation (RAEB-T). The starting doseof 3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione was25 mg daily for the first 13 patients and 10 mg daily for the remainingthree patients. All patients receiving the starting dose of 25 mgrequired dose reduction by the completion of eight weeks therapy. Amongthese 16 patients who completed at least 8 weeks of monitoring, ninepatients achieved an erythroid response as assessed by the InternationalMDS Working Group Criteria. The erythroid responses consisted oftransfusion independence in seven previously transfusion-dependentpatients, a >2 g/dL rise in blood hemoglobin concentration in onepatient in with transfusion-independent anemia, and a >50% decrease inRBC transfusion requirement in one transfusion-dependent patient.Therefore, a major erythroid response developed in eight of 16 patientsand a minor erythroid response was observed in one patient. All of ninepatients who showed erythroid response were low- or intermediate-1-riskpatients. One patient also had a minor platelet response. In addition,complete cytogenetic responses developed in five in eight patients withabnormal karyotypes at baseline. These five patients with completecytogenetic responses all had the Del5q31-33 abnormality, which has beendiscovered to be a good prognostic factor for MDS. Indeed, all fivepatients who enrolled in this study with 5q-syndrome achieved a completecytogenetic response and a major erythroid response. The study alsoindicated an association of this therapy with an increased apoptoticindex for myelodysplastic progenitors and recovery of normalhematopoietic progenitor cells.

5.4. Cycling Therapy in MDS Patients

As mentioned above, immunomodulatory compounds of the invention can becyclically administered to patients with MDS. Cycling therapy involvesthe administration of a first agent for a period of time, followed bythe administration of the agent and/or the second agent for a period oftime and repeating this sequential administration. Cycling therapy canreduce the development of resistance to one or more of the therapies,avoid or reduce the side effects of one of the therapies, and/orimproves the efficacy of the treatment.

Example 1

In a specific embodiment, prophylactic or therapeutic agents areadministered in a cycle of about 16 weeks, about once or twice everyday. One cycle can comprise the administration of a therapeutic onprophylactic agent and at least one (1), two (2), or three (3) weeks ofrest. The number of cycles administered is from about 1 to about 12cycles, more typically from about 2 to about 10 cycles, and moretypically from about 2 to about 8 cycles.

Example 2

The objectives of the study are to evaluate the efficacy and safety oforal administration of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione inpatients with MDS. Patients receive the compound in an amount of 10 mg/dor 15 mg/d for 21 days every 28 days in 4-week cycles for 16 weeks (4cycles) or 24 weeks (6 cycles). The subject population comprisespatients with low- or intermediate-1-risk MDS (International PrognosticScoring System) with red blood cell transfusion-dependent anemia whohave received at least two units of RBCs within 8 week of baseline(first day of study treatment). In addition to hematological laboratorymonitoring, bone marrow aspirates/biopsies with cytogenic analyses areobtained at baseline, after the completion of 3 cycles and after thecompletion of 6 cycles. The bone marrow, safety and efficacy data arereviewed to assess benefit-to-risk considerations throughout the study.The study reviews red blood cell transfusion independence and majorerythroid response according to the International MDS Working GroupCriteria. Further, the study observes red blood cell transfusionindependence in the subgroup of patients with the 5q deletioncytogenetic abnormality; platelet, neutrophil, bone marrow andcytogenetic responses; and minor erythroid response of ≧50% but <100%reduction in red blood cell transfusion requirement over an 8 weekperiod. The study further monitors adverse events, hematological tests,serum chemistries, TSH, urinalysis, urine or serum pregnancy tests,vital signs, ECG and physical examinations.

Example 3

The objectives of the study are to compare the efficacy and safety oforal administration of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione to thatof placebo plus standard care in patients with MDS. Patients receive thetherapy in 4-week cycles for 16 weeks (4 cycles) or 24 weeks (6 cycles).The subject population comprise patients with low- orintermediate-1-risk MDS (International Prognostic Scoring System) withred blood cell transfusion-dependent anemia who have received at leasttwo units of RBCs within 8 week of baseline (first day of studytreatment). The study visits to assess safety and efficacy occur every 4weeks and hematologic laboratory monitoring is performed every 2 weeks.Bone marrow aspirates/biopsies with cytogenetic analyses are obtained atbaseline after the completion of 3 cycles and after the completion of 6cycles. Bone marrow findings, safety and efficacy data are reviewed toassess benefit-to-risk considerations throughout the study. An extensionstudy of continued treatments with the administration of the compound isavailable for patients who derive clinical benefit from 6 cycles of thetherapy and to provide an opportunity for subjects who were randomizedto placebo to cross over to the therapy.

Embodiments of the invention described herein are only a sampling of thescope of the invention. The full scope of the invention is betterunderstood with reference to the attached claims.

What is claimed is:
 1. A method of treating a myelodysplastic syndrome,which comprises (a) administering to a patient in need thereof about 0.1mg per day to about 10 mg per day of4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione of formula:

or a pharmaceutically acceptable salt, solvate or stereoisomer thereoffor a period of time followed by a period of rest; and (b) repeatingstep (a).
 2. The method of claim 1, wherein the compound is apharmaceutically acceptable salt.
 3. The method of claim 1, wherein thecompound is a pharmaceutically acceptable solvate.
 4. The method ofclaim 1, wherein the stereoisomer is an enantiomerically pure R isomeror S isomer.
 5. The method of claim 1, which further comprises (c)administering a therapeutically effective amount of a second activeagent for a period of time followed by a period of rest; and (d)repeating step (c).
 6. The method of claim 5, wherein the second activeagent is capable of improving blood cell production.
 7. The method ofclaim 5, wherein the second active agent is a cytokine, hematopoieticgrowth factor, an anti-cancer agent, an antibiotic, a proteasomeinhibitor, or an immunosuppressive agent.
 8. The method of claim 5,wherein the second active agent is etanercept, imatinib, anti-TNF-αantibodies, infliximab, G-CSF, GM-CSF, EPO, topotecan, pentoxifylline,ciprofloxacin, irinotecan, vinblastine, dexamethasone, IL2, IL8, IL18,Ara-C, vinorelbine, isotretinoin, 13-cis-retinoic acid, arsenictrioxide, or a pharmacologically active mutant or derivative thereof. 9.The method of claim 1, wherein the myelodysplastic syndrome isrefractory anemia, refractory anemia with ringed sideroblasts,refractory anemia with excess blasts, refractory anemia with excessblasts in transformation, or chronic myelomonocytic leukemia.
 10. Themethod of claim 1, wherein the compound is administered orally.
 11. Themethod of claim 10, wherein the compound is administered in the form ofa capsule or tablet.
 12. The method of claim 1, wherein the number ofcycle is from one to twelve cycles.
 13. The method of claim 1, thecompound is administered in an amount of from about 0.1 to about 10 mgper day for 21 days every 28 days for sixteen or twenty-four weeks. 14.The method of claim 1, wherein the compound is administered in an amountof from about 0.1 mg per day to about 10 mg per day for 21 days followedby seven days of rest in a 28 days cycle.
 15. The method of claim 1,wherein the compound is administered orally in an amount of 0.1 mg, 1mg, 2 mg, 2.5 mg, 5 mg or 10 mg as a capsule per day.
 16. The method ofclaim 1, wherein the4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione isadministered.
 17. The method of claim 5, wherein the second active agentis azacytidine.